In vitro/in vivo variation in osteogenic gene expression and 1,25-dihydroxyvitamin D3 response of different murine osteosarcomas. Three unique clonal murine osteosarcoma lines (K7, K8, and K14) were grown in vitro under conditions permissive for mineralization or as subcutaneous implants in syngeneic mice. In vitro, K8 cells exhibited the most mineralization, followed by K14, then by K7. K8 cells constitutively expressed bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (Apase), osteocalcin (OC), and Type I collagen (COLlAl) at the highest levels. Subconfluent K14 cells showed expression of the osteogenic phenotype, including OC, with down-regulation after several days in culture. In contrast, K7 cells showed a gradual up-regulation of these genes, with the exception of OC. 1 ,25-dihydroxyvitamin D3 ( 1 ,25-D3) stimulated expression of OC in K14 cells, was weakly inhibitory in K8 cells, and had no effect on K7 cells. In contrast to its variable effect on OC expression, 1.25-D3 stimulated OPN expression in both the K8 and K14 cells. In vivo, K8 cells produced extensive mineralized woven bone after subcutaneous implantation, with complete absence of osteoclasts, while K14 and K7 tissues were less ossified. In vivo, all lines expressed all bone-specific genes with the exception of OC which was expressed at high levels only by the K14 cells. We infer that factor(s) other than 1,25-D3 stimulate K7 differentiation in vivo and that the molecular mechanisms by which 1,25-D3 affects specific gene regulation may occur via interaction of secondary factor(s) with the 1,25-D3 receptor. The in vitro/in vivo data indicate that K8 are constitutive osteogenic cells that appear uncoupled from systemic and local tissue regulation. K14 are conditionally osteogenic, capable of up-regulation of their osteogenic phenotype in host mice and in vitro by 1,25-D3. K7 are also conditionally osteogenic, stimulated to differentiate in vivo, but uncoupled from 1,25-D3 regulation in vitro.